Molecular mechanism for the synchronized electrostatic coacervation and co-aggregation of alpha-synuclein and tau

Amyloid aggregation of α-synuclein (αS) is the hallmark of Parkinson’s disease and other synucleinopathies. Recently, Tau protein, generally associated with Alzheimer’s disease, has been linked to αS pathology and observed to co-localize in αS-rich disease inclusions, although the molecular mechanisms for the co-aggregation of both proteins remain elusive. We report here that αS phase-separates into liquid condensates by electrostatic complex coacervation with positively charged polypeptides such as Tau. Condensates undergo either fast gelation or coalescence followed by slow amyloid aggregation depending on the affinity of αS for the poly-cation and the rate of valence exhaustion of the condensate network. By combining a set of advanced biophysical techniques, we have been able to characterize αS/Tau liquid-liquid phase separation and identified key factors that lead to the formation of hetero-aggregates containing both proteins in the interior of the liquid protein condensates.


Aggregation assays
ThT-monitored aggregation assays were performed as described by Sandberg & Nyström 1 . 50 µM of Tau441, AggDef-Tau or K18 monomer were incubated in PB buffer pH 7.4, 25 mM NaCl, 25 µM ThT, 0.02 % azide, in the presence of 12.5 µM of heparin (Sigma-Aldrich) at 37 °C and under shaking conditions (700 rpm using in situ orbital agitation in the plate reader) until reaction was complete. Non-Binding 96-Well Microplate (µClear®, Black, F-Bottom/Chimney Well), Greiner bio-one North America Inc., USA) were used and the plates were covered with adhesive foil to prevent evaporation. All buffer samples and additive stock solutions were prefiltered with 0.22 mm filters and the multi-well plates were thoroughly cleaned before use. Kinetic reads were recorded in a FLUOstar plate reader (BMG Labtech, Germany) with excitation at 450 ± 5 nm and emission at 485 ± 5 nm.
Atomic force microscopy 20 µL of Tau441 aggregates resulting from heparin-induced aggregation as described above and αS/Tau441 isolated puncta from 24 h samples as described in Liquid-to-solid phase transition (LSPT) puncta isolation (Methods) were deposited on freshly cleaved Muscovite Mica V-5 (Electron Microscopy Sciences; Hatfield, Pensilvania, USA). Slides were washed three times with more than 5 mL of double distilled water to remove PEG and salts and allowed to dry before imaging acquisition on a Bruker Multimode 8 (Bruker; Billerica, USA) using a FMG01 gold probe (NT-MDT Spectrum Instruments Ltd., Russia) in intermittent-contact mode in air. Images were processed using Gwyddion 2.56 and the width measurements were corrected for the tip shape and size (10 nm).

Supplementary Figures
Supplementary Figure 1. Spatial distribution of S, Tau441, N t -Tau and macromolecular crowders in the coacervate samples. Representative confocal fluorescence (CF) microscopy images of colocalizing S/Tau441 (a, 10 µM each protein) and S/N t -Tau (b, 25 µM each) coacervates. The intensity profiles obtained at the center of the image (shown as discontinuous lines in the center of the merge image) are shown for the fluorescence intensity of S-AF488 (green) and Tau441-or N t -Tau-Atto647N (red). c) Representative WF microscopy images of S/Tau441 coacervates (25 µM each) in 20 % dextran-70 (w/v). d) Representative CF microscopy images of S/Tau441 LLPS in different crowders. In green, the fluorescence intensity of FITC-labeled crowders (1 % with respect to the total crowder concentration) is shown and, in red, Atto647N-labeled S (1 µM). For the coacervate samples in the presence of PEG-8, S and Tau were used at 10 µM each protein and PEG at 15 % (w/v), while for the coacervate samples in the presence of dextran-70, the proteins were used at 25 µM each, and the crowder at 20 % (w/v). The dotted white rectangle indicates the analyzed area used to obtain the fluorescence intensity profiles shown in the right panels. Scale bars are 5 µm in panels a-c and 10 µm in panel d. Experiments were performed in triplicate with comparable results. Figure 2. Role of electrostatic interactions and quantification of the coacervation of S with the poly-cations. a) Representative WF (left) and BF (right) microscopy images of the effect of 1 M NaCl and 10 % (v/v) 1,6-Hexanediol (1,6-HD) on S/Tau441 coacervates (10 µM each protein, 1 µM AF488-S and Atto647N-Tau441 for WF microscopy). Scale bar = 20 µm. b) Light scattering (at 350 nm) of Tau441 and S/Tau441 coacervate samples (10 µM each protein) in the absence or the presence of 1 M NaCl or 10 % 1,6-HD (N=2-3 sample replicas, as indicated). c) Representative WF microscopy images of αS/ΔN t -Tau coacervates (50 μM each protein) formed in the presence of 5 % PEG, in the absence (left) or presence (right) of 10 % (v/v) 1,6-HD. Scale bar is 20 µm. d) Light scattering (at 350 nm) of S or C t -S with N t -Tau or K18 (50 µM each protein) in LLPS buffer (15 % PEG) (N=2-3 sample replicas, as indicated). Samples without proteins served as control. Samples with only N t -Tau or K18 in LLPS buffer are indicated as -LLPS samples for each protein data set, as no droplets are observed. e) Quantification of the fraction of S in the dispersed phase in the different LLPS systems: 100 µM S with 1 mM pLK or 100 µM Tau441 or N t -Tau (100% was taken from the samples of S in the absence of poly-cation in each independent gel). The LLPS samples were centrifuged after 30 min incubation and the fraction of S remaining in the disperse phase (f S,d ) was determined by SDS-PAGE gel analysis. The protein bands were resolved by coomassie staining for the S/pLK system and by fluorescence for the S/Tau441 and S/N t -Tau systems (for both S and Tau variants quantification -1 µM of fluorescently-labeled proteins-although only S quantification is shown). D1 and D2 indicate independent duplicate experiments. Each individual gel was analyzed independently. Source data with the unprocessed gel images are provided as a Source Data file. % PEG with (blue) or without (red) 10-molar equivalents of pLK. Best simulations are shown in dark blue and dark red for labeled S with or without pLK, respectively. The EPR signal of the protein in the dispersed phase is consistent with a nitroxide radical in the fast motion regime, characterized by g iso = 2.0055 and axial hyperfine coupling (A = [20 20 104] MHz). For the spectra of TEMPOL-24-S, a one-component isotropic model with virtually identical correlation times for the sample with and without pLK was obtained (corr. time 6.5•10 -10 s). For TEMPOL-122-S, the sample in absence of pLK could be well simulated to one-component isotropic model with a corr. time 7.5•10 -10 s, while two-component isotropic model was needed to simulate the data for the sample with pLK (30% with a corr. time of 7.5•10 -10 s and 70% with a slower component with corr. Time 2.0•10 -9 s). Residuals were calculated as described in the "Methods" section and are shown for labeled S with (dark blue) or without (dark red) pLK. c) Secondary chemical shifts and (d) R 1ρ relaxation analysis for 150 µM 13 C/ 15 N-labeled S in the presence of 1.5 mM pLK (green), 75 M N t -Tau (light blue) or 225 M N t -Tau (dark blue), respectively. Data and error bars correspond to the R 1 rates and experimental uncertainties obtained from fitting the intensities of the peaks over the distinct time delays to an exponential function. The data show the reduction of conformational flexibility in the C-terminal region of S upon interacting with poly-cations. Source data are provided as a Source Data file.

Supplementary Figure 6. EPR binding titration of S and different poly-cations.
Normalized CW X-Band EPR spectra (top) and zooms (bottom) of 50 µM TEMPOL-122-S in 15 % PEG-8 in the presence of increasing concentrations of Tau441 (left), N t -Tau (center) or pLK (right). A cold-to-hot color code indicates increasing concentrations of the poly-cation. The second and third bands of the spin probe spectrum, used for the titration analysis (see Fig. 3e and Methods for more information) are indicated as I II and I III, respectively, where I stands for intensity . Source data are provided as a Source Data file.

Supplementary Figure 7. Coalescence and wetting properties of S/Tau441 coacervates. a)
Representative WF microscopy images of S/Tau441 coacervates (25 M each protein, 1 M AF488-labeled S) visualized at the bottom of the plate wells (z = 0 m) after 24 h incubation using microwell plates with different material coatings as indicated. Both non-binding plates (PEG-based coated microwell plates typically used as non-binding plates for hydrophobic protein samples) and glass plates have hydrophilic coatings. As hydrophobic material, nontreated 96-well polystyrene microplates were used. b) A fusion event of two S/Tau441 rafts as observed by BF microscopy is shown. Scale bars are 20 m. c) Light scattering signal (at 350 nm) of an S/ Tau441  The images show the same microscopy field with a large raft composed of both proteins occupying the entire field (shown in green in both images -note that the background intensity is ~ 20 counts) in which several puncta are visualized as a condensation of proteins (higher intensity values, visualized as brighter green-yellow-red spots). This correlates with lower fluorescence lifetimes (due to condensation-induced self-quenching) visualized as blue spots that coincide with the more intense spots. Data is shown for the Atto647N channel. Scale bars = 10 m. b-c) Analytical approach for species-specific lifetime analysis. A representative lifetime colorcoded image of S/Tau441 coacervates after 24 h incubation is shown in b, and the selection of regions of interest (ROIs) containing species-specific lifetimes (ROI puncta , ROI raft ) are selected from the overall image (ROI all ). Data are shown for the Atto647N channel. Scale bars are 5 m. c) Representative normalized lifetime decays from species-specific ROIs for the AF488 (left) and Atto647N (right) channels. Source data are provided as a Source Data file. d) Representative lifetime color-coded FLIM images (left panels) of S/N t -Tau droplets at initial times and after 24 h incubation and deposited on the bottom of the well (25 M each protein, 1 M AF488-S and Atto647N-N t -Tau; scale bar = 20 m) and their pixel-wise lifetime analysis (right, box plot). 24h-incubated gelated droplets (D g ) are shown in light turquoise blue (N = 10). Early droplets (D) and puncta (P) are the same as in Fig. 6c but are shown here for comparison. Source data are provided as a Source Data file. Mean and median values are shown as yellow squares and black lines within the boxes, respectively. Lower and upper box limits indicate the first and third quartile, respectively, while minimum and maximum values within 1.5 x interquartile range (IQR) are shown as whiskers. Outliers are shown as black diamonds. The statistical significance between pairs of distributions was determined with a two sample t-test assuming unequal variances. No significant differences were found for droplets that gelated after 24-h incubation (D g ). The p-value from a two-tailed t-test is shown as stars for each compared pair of data (* p-value > 0.01, ** p-value > 0.001, *** p-value > 0.0001, **** p-value > 0.00001, ns means not significant (p-value > 0.05). Precise p-values are provided in Supp. Table 1. e) Lifetime color-coded FLIM images showing the heterogeneity of size, shape and fluorescence lifetime in puncta from Tau441, S/Tau441 and S/N t -Tau. For S/Tau441 and S/N t -Tau puncta the variability in S content is also evident. Scale bar = 1 m and 24 h (right). Higher, "red-shifted", E values in puncta (white arrows) with respect to the droplets (white circles) and rafts (white arrowheads) indicate LSPT. Scale bars are 5 m for the left images and 2 m for the center and right images. b) Normalized FRET efficiency histograms for the different protein structures observed (droplets, rafts and puncta) in S/Tau441 (top) and S/N t -Tau (bottom) LLPS and LSPT processes. Each histogram is composed of a triplicate experiment with 2 analyzed fields per sample. Source data are provided as a Source Data file.